Experimental validation and complexity of miRNA-mRNA target interaction during zebrafish primitive erythropoiesis

Biochem Biophys Res Commun. 2009 Apr 17;381(4):688-93. doi: 10.1016/j.bbrc.2009.02.122. Epub 2009 Feb 28.


MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. Despite increasing number of genes has been predicted to be miRNA targets by bioinformatics analysis and luciferase-based reporter assay in vitro (RA-In Vitro), few of them have been experimentally validated in physiological context. Using transient reporter assay in vivo (TRA-In Vivo), we identified hydroxymethylbilane synthase b (hmbsb) and Krüppel-like transcription factor d (klfd) as potential target gene for miR-451 and miR-144, respectively. Although hmbsb, miR-451, klfd and miR-144 are all co-expressed in the developing erythroid progenitors during zebrafish erythropoiesis, only klfd can be validated as a bona fide physiological target of miR-144 using a transgene-based physiological reporter assay in vivo (PRA-In Vivo). Thus, failure to verify hmbsb as miR-451 target in physiological context raises a crucial question as to how to determine bona fide target of miRNA. The results address the importance of using multiple approaches combined with Western blot analysis to validate the physiological target of a given miRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Erythroid Cells / enzymology
  • Erythropoiesis / genetics*
  • Evolution, Molecular
  • Gene Expression Regulation, Developmental
  • Gene Expression Regulation, Enzymologic
  • Genes, Reporter
  • Heme / biosynthesis
  • Hydroxymethylbilane Synthase / genetics*
  • Kruppel-Like Transcription Factors / genetics*
  • MicroRNAs / metabolism*
  • RNA, Messenger / metabolism*
  • Zebrafish / embryology*
  • Zebrafish / genetics
  • Zebrafish / physiology
  • Zebrafish Proteins / genetics*


  • Kruppel-Like Transcription Factors
  • MicroRNAs
  • Mirn144 microRNA, zebrafish
  • Mirn451 microRNA, zebrafish
  • RNA, Messenger
  • Zebrafish Proteins
  • klf1 protein, zebrafish
  • Heme
  • Hydroxymethylbilane Synthase